Background: Metformin inhibits breast cancer cell proliferation and colony formation, with S phase arrest and the induction of apoptosis in triple negative breast cancer cells in vitro. In these cells, metformin down-regulates Cyclin D1 and Cyclin E, as well as EGFR, p-EGFR, p-AKT, p-MAPK, p-Src and p-mTOR, whereas it up-regulates p-AMPK (Liu, et al. Cell Cycle, 2009). Non-triple negative breast cancer cells are resistant to metformin induced apoptosis, although they show similar changes in p-AMPK induction with metformin (Alimova, et al. Cell Cycle, 2009). We hypothesized that there are unique signaling intermediates associated with metformin responsivity in triple negative cells and have identified the signal transducer and activator of transcription 3 (Stat3) as a potential target. Stat3 is constitutively activated in a wide range of tumors, including breast cancer (up to 60%). It reportedly promotes cancer cell proliferation and survival. Methods: Human triple negative breast cancer cell lines (MDA-468, MDA-231, BT20 and HCC70) were used to evaluate interactions between metformin and Stat3 signaling. Activation of Stat3 was examined by Western blot analysis with phosphorylation-specific antibodies. Cell proliferation was determined by MTS assay. Apoptosis was quantitated by an apoptosis ELISA assay and Western blots for PARP and caspase cleavage. In these 4 cell lines, Stat3-over-expressing clones were obtained via transfection of a constitutive active (CA) construct of Stat3. Specific knock-down of Stat3 expression was achieved by using a lentiviral system containing Stat3 small hairpin RNA. Results: In a dose and time dependent manner, metformin inhibits Stat3 phosphorylation at sites Tyr705 and Ser727, Cyclin D1 and E protein expression, cleavage of PARP and the pro-caspases 3, 8, and 9. Overexpression of the CA-Stat3 attenuates the aforementioned meformin-associated PARP and caspase cleavage as well as apoptosis, and it suppresses metformin induced cell cycle arrest and changes in cyclin D1 and E. In contrast, specific knock-down of Stat3 expression sensitizes the triple negative breast cancer cells to metformin-mediated apoptosis and cell cycle arrest, enhancing the signaling changes we describe above. Conclusion: These data indicate that Stat3 is a critical intermediary for metformin action in triple negative breast cancer cells. Our studies suggest that targeting Stat3 activation may be a useful strategy to treat breast cancer patients with triple negative phenotype. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD03-09.

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